Pharmaceutical compositions comprising forms of 5-azacytidine

ABSTRACT

The invention provides novel polymorphic and pseudopolymorphic crystalline forms of 5-azacytidine, along with methods for preparing said forms, wherein 5-azacytidine is represented by the formula: 
     
       
         
         
             
             
         
       
     
     The invention also includes pharmaceutical compositions comprising said forms.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/787,214, filed May 25, 2010, which is a continuation of U.S. Pat. No.7,772,199, filed Jul. 18, 2006, entitled, “Forms of 5-Azacytidine,”which is a continuation of U.S. Pat. No. 7,078,518, filed Feb. 7, 2005,entitled, “Forms of 5-Azacytidine,” which is a divisional of U.S. Pat.No. 6,887,855, filed Mar. 17, 2003, entitled “Forms of 5-Azacytidine,”the contents of all of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The invention relates to polymorphic and pseudopolymorphic forms of5-azacytidine (also known as azacitidine and4-amino-1-β-D-ribofuranosyl-S-triazin-2(1H)-one), methods for preparingsaid forms, and pharmaceutical compositions comprising said forms.5-azacytidine may be used in the treatment of disease, including thetreatment of myelodysplastic syndromes (MDS).

BACKGROUND OF THE INVENTION

Polymorphs exist as two or more crystalline phases that have differentarrangements and/or different conformations of the molecule in a crystallattice. When a solvent molecule(s) is contained within the crystallattice the resulting crystal is called a pseudopolymorph, or solvate.If the solvent molecule(s) within the crystal structure is a watermolecule, then the pseudopolymorph/solvate is called a hydrate. Thepolymorphic and pseudopolymorphic solids display different physicalproperties, including those due to packing, and various thermodynamic,spectroscopic, interfacial and mechanical properties (See H. Brittain,Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, N.Y.,1999, pp. 1-2). Polymorphic and pseudopolymorphic forms of the drugsubstance (also known as the “active pharmaceutical ingredient” (API)),as administered by itself or formulated as a drug product (also known asthe final or finished dosage form, or as the pharmaceutical composition)are well known and may affect, for example, the solubility, stability,flowability, fractability, and compressibility of drug substances andthe safety and efficacy of drug products, (see, e.g., Knapman, K ModemDrug Discoveries, March 2000: 53).

5-azacytidine (also known as azacitidine and4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one; Nation ServiceCenter designation NSC-102816; CAS Registry Number 320-67-2) hasundergone NCI-sponsored clinical trials for the treatment ofmyelodysplastic syndromes (MDS). See Kornblith et al., J. Clin. Oncol.20(10): 2441-2452 (2002) and Silverman et al., J. Clin. Oncol. 20(10):2429-2440 (2002). 5-azacytidine may be defined as having a formula ofC₈H₁₂N₄O₅, a molecular weight of 244.20 and a structure of:

The polymorphic form of 5-azacytidine drug substance and drug producthas never been characterized. It is an object of the present inventionto characterize the polymorphic forms of 5-azacytidine.

SUMMARY OF THE INVENTION

It has been unexpectedly found that 5-azacytidine exists in at leasteight different polymorphic and pseudopolymorphic crystalline forms(Forms I-VIII), in addition to an amorphous form. Form I is a polymorphfound in prior art retained samples of 5-azacytidine drug substance.Form II is a polymorph found in some prior art retained samples of the5-azacytidine drug substance; in those samples, Form II is always foundin mixed phase with Form I. Form III is a hydrate, and is formed whenprior art retained and current samples of the drug product arereconstituted with water to form a “slurry” prior to administration tothe patient. Form VI is found in prior art retained samples of the5-azacytidine drug product, either substantially free of otherpolymorphs, or in mixed phase with Form I.

The invention provides novel crystalline forms referred to as Form IV,Form V, Form VII and Form VIII. Forms I-VIII each have characteristicX-ray power diffraction (XRPD) patterns and are easily distinguishedfrom one another using XRPD.

Also included in the present invention are methods for robustly andreproducibly synthesizing 5-azacytidine drug substance substantially asForm IV, Form V, or Form VIII. Also provided are methods for robustlyand reproducibly synthesizing a Form I/VII mixed phase. The inventionalso provides pharmaceutical compositions comprising the various formsof 5-azacytidine together with one or more pharmaceutically acceptableexcipients, diluents, or carriers.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the X-Ray Powder Diffraction (XRPD) pattern of5-azacytidine, Form I, labeled with the most prominent 2θ angles (Cu Kαradiation).

FIG. 2 presents the XRPD pattern of 5-azacytidine, mixed phase Form Iand Form II, labeled with the most prominent 2θ angles (Cu Kαradiation).

FIG. 3 presents the XRPD pattern of 5-azacytidine, Form III, labeledwith the most prominent 2θ angles (Cu Kα radiation).

FIG. 4 presents the XRPD pattern of 5-azacytidine, Form IV, labeled withthe most prominent 2θ angles (Cu Kα radiation).

FIG. 5 presents the XRPD pattern of 5-azacytidine, Form V, labeled withthe most prominent 2θ angles (Cu Kα radiation).

FIG. 6 presents the XRPD pattern of 5-azacytidine, Form VI, labeled withthe most prominent 2θ angles (Cu Kα radiation).

FIG. 7 presents the XRPD pattern of 5-azacytidine, mixed phase Form Iand Form VII, labeled with the most prominent 2θ angles (Cu Kαradiation).

FIG. 8 presents the XRPD pattern of 5-azacytidine, Form VIII, labeledwith the most prominent 2θ angles (Cu Kα radiation).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 5-azacytidineCrystalline Forms I-VIII

It has been discovered that 5-azacytidine exists in at least eightdifferent polymorphic and pseudopolymorphic crystalline forms, and alsoin an amorphous form.

Form I

A single sample of the 5-azacytidine drug substance was synthesized from5-azacytosine and 1,2,3,5,-Tetra-O-acetyl-β-D-ribofuranose according tothe prior art method provided in Example 1. The last step of this methodis a recrystallization of the crude synthesis product from aDMSO/methanol co-solvent system. Specifically, the crude synthesisproduct is dissolved in DMSO (preheated to about 90° C.), and thenmethanol is added to the DMSO solution. The co-solvent mixture isequilibrated at approximately −20° C. to allow 5-azacytidine crystalformation. The product is collected by vacuum filtration and allowed toair thy.

The X-Ray Powder Diffraction (XRPD: see Example 5) pattern of theresulting 5-azacytidine is shown in FIG. 1 along with some of the 2θvalues. Table 1 provides the most prominent 2θ angles, d-spacing andrelative intensities for this material, which is designated as Form I.

TABLE 1 5-azacytidine Form I - the most prominent 2θ angles, d-spacingand relative intensities (Cu Kα radiation) 2θ Angle (°) d-spacing (Å)Relative Intensity 12.182 7.260 39.1 13.024 6.792 44.1 14.399 6.146 31.516.470 5.378 27.1 18.627 4.760 16.0 19.049 4.655 35.9 20.182 4.396 37.021.329 4.162 12.4 23.033 3.858 100.0 23.872 3.724 28.0 26.863 3.316 10.827.135 3.284 51.5 29.277 3.048 25.6 29.591 3.016 11.5 30.369 2.941 10.832.072 2.788 13.4

Thermal analysis of Form I indicates that this form of 5-azacytidine isanhydrous. See Example 6.

Form II

Retained samples of the drug substance previously used to formulate thedrug product in the NCI-sponsored Cancer and Leukemia Group B (CALGB)investigations (Phase 2 trial 8291 and Phase 3 trial 9221) for thetreatment of MDS (Investigational New Drug (IND) 7574) were alsoanalyzed by XRPD. The retained drug substance samples comprised eitherForm I, or a mixed phase of Form I and another polymorph: Form II. SeeExample 5.

The XRPD powder pattern of mixed phase Forms I and II is shown in FIG. 2along with some of the 2θ values. Peaks distinctive to Form II areobserved at 13.5, 17.6 and 22.3° 2θ. Table 2 provides the most prominent2θ angles, d-spacing and relative intensities for this mixed phase.

TABLE 2 5-azacytidine, Mixed Phase Forms I and II - the most prominent2θ angles, d-spacing and relative intensities (Cu Kα radiation) 2θ Angle(°) d-spacing (Å) Relative Intensity 12.244 7.223 34.8 13.082 6.762 37.013.458* 6.574 29.2 14.452 6.124 25.4 16.521 5.361 19.0 17.648* 5.02212.1 18.677 4.747 12.7 19.093 4.645 41.3 20.231 4.386 42.1 21.353 4.15815.5 22.309* 3.982 35.1 23.070 3.852 100.0 23.909 3.719 18.9 26.6413.343 18.2 26.813 3.322 12.6 27.158 3.281 46.0 29.309 3.045 27.3 29.6093.015 12.7 30.384 2.939 10.5 32.074 2.788 12.0

These results indicate that the prior art 5-azacytidine synthesisprocedures for the drug substance produce either Form I substantiallyfree of other forms, or a Form I/II mixed phase i.e. a solid material inwhich 5-azacytidine is present in a mixed phase of both Form I and FormII.

Thermal analysis of mixed phase Form I/II is presented in Example 6.

Form III

An additional crystalline form of 5-azacytidine, designated Form III, isfound in slurries of 5-azacytidine. See Example 8. Moreover, it has beenfound that all forms of 5-azacytidine (including the 5-azacytidine inthe prior art drug product) convert to Form III in water. See Example 8.Thus, reconstitution of the drug product used in the aforementioned NCItrials would have led to the formation of a saturated solution (or“slurry”) in which the remaining solid 5-azacytidine was Form III. TheXRPD powder pattern of Form III is shown in FIG. 3 along with some ofthe 2θ values. Table 3 provides the most prominent 2θ angles, d-spacingand relative intensities for this crystalline material. The XRPD powderpattern for Form III is distinctly different from that of all of theother forms of 5-azacytidine.

TABLE 3 5-azacytidine, Form III - the most prominent 2θ angles,d-spacing and relative intensities (Cu Kα radiation) 2θ Angle (°)d-spacing (Å) Relative Intensity 6.566 13.450 32.9 11.983 7.380 52.513.089 6.758 71.0 15.138 5.848 38.9 17.446 5.079 48.2 20.762 4.275 10.821.049 4.147 34.8 22.776 3.901 89.5 24.363 3.651 13.7 25.743 3.458 22.826.305 3.385 39.9 28.741 3.104 100.0 31.393 2.847 22.5 32.806 2.728 11.833.043 2.709 10.1 33.536 2.670 15.1 36.371 2.468 11.0 39.157 2.299 19.341.643 2.167 12.1

Thermal analysis and proton (¹H) NMR spectroscopy indicate that Form IIIis a pseudopolymorphic form of 5-azacytidine, specifically amonohydrate. See Examples 6-7.

Form IV

Form IV is a novel crystalline form of 5-azacytidine. Form IV wasrecovered by slow recrystallization from a DMSO/toluene co-solventsystem (see Example 2) or by fast recrystallization from theDMSO/chloroform co-solvent system (see Example 3). The XRPD powderpattern of Form IV is shown in FIG. 4 along with some of the 2θ values.Table 4 provides the most prominent 2θ angles, d-spacing and relativeintensities for this crystalline material. The XRPD powder pattern forForm IV is distinctly different from that of any other form.

TABLE 4 5-azacytidine Form IV - the most prominent 2θ angles, d-spacingand relative intensities (Cu Kα radiation) 2θ Angle (°) d-spacing (Å)Relative Intensity 5.704 15.408 24.9 11.571 7.642 97.8 12.563 7.040 22.214.070 6.289 100.0 15.943 5.555 67.4 16.993 5.213 51.0 18.066 4.906 20.120.377 4.355 44.7 20.729 4.281 49.0 21.484 4.132 36.30 21.803 4.073 11.222.452 3.957 66.7 22.709 3.913 64.0 23.646 3.760 17.3 24.068 3.695 19.425.346 3.526 12.0 25.346 3.511 12.5 26.900 3.312 11.0 27.991 3.185 11.428.527 3.126 25.7 28.723 3.106 34.1 30.124 2.964 14.7 30.673 2.912 53.631.059 2.877 15.7 35.059 2.557 18.1 38.195 2.354 15.0 38.403 2.342 12.6

Thermal analysis of Form IV is presented in Example 6.

Form V

Form V is a novel crystalline form of 5-azacytidine. Form V wasrecovered by fast recrystallization of 5-azacytidine from a DMSO/tolueneco-solvent system (see Example 3). The XRPD powder pattern of Form V isshown in FIG. 5 along with some of the 2θ values. Table 5 provides themost prominent 2θ angles, d-spacing and relative intensities for thiscrystalline material. The XRPD powder pattern for Form V is distinctlydifferent from that of any other form.

TABLE 5 5-azacytidine Form V - the most prominent 2θ angles, d-spacingand relative intensities (Cu Kα radiation) 2θ Angle (°) d-spacing (Å)Relative Intensity 11.018 8.024 40.0 12.351 7.160 29.6 13.176 6.714 28.313.747 6.436 42.9 14.548 6.084 18.3 15.542 5.697 14.2 16.556 5.350 47.817.978 4.930 18.1 18.549 4.780 83.9 19.202 4.618 25.0 19.819 4.476 12.120.329 4.365 28.6 21.518 4.126 100.0 21.970 4.042 65.6 22.521 3.948 11.523.179 3.834 66.5 24.018 3.702 13.0 24.569 3.620 40.7 27.224 3.273 50.228.469 3.133 24.2 29.041 3.072 24.8 29.429 3.033 15.0 30.924 2.889 15.631.133 2.870 22.6 37.938 2.370 10.7

Thermal analysis indicates that Form V is a solvate. See Example 6.

Form VI

The drug product used in the aforementioned NCI investigation wastypically prepared by lyophilizing a solution of 5-azacytidine andmannitol (1:1 w/w). The resultant drug product comprised 100 mg of5-azacytidine and 100 mg mannitol as a lyophilized cake in a vial andwas administered by subcutaneous injection as an aqueous suspension(“slurry”). XRPD analysis of retained samples of the drug product usedin the NCI investigation revealed the existence of another polymorph,Form VI. The retained drug product samples comprised either Form VIalone, or a Form I/VI mixed phase. Table 6 provides the most prominent2θ angles, d-spacing and relative intensities for Form VI.

TABLE 6 5-azacytidine Form VI - the most prominent 2θ angles, d-spacingand relative intensities (Cu Kα radiation) 2θ Angle (°) d-spacing (Å)Relative Intensity 12.533 7.057 10.1 12.963 6.824 10.2 13.801 6.411100.0 18.929 4.6843 10.0 20.920 4.243 34.2 21.108 4.205 49.4 21.5274.125 47.0 22.623 3.922 10.7 22.970 3.869 13.8 24.054 3.697 77.8 26.6683.340 23.0 27.210 3.275 33.7 28.519 3.127 12.9 29.548 3.021 27.2 30.4582.932 50.3 33.810 2.649 11.6 35.079 2.556 12.6 37.528 2.411 24.7

Thermal analysis and proton (¹H) NMR spectroscopy of Form VI ispresented in Examples 6-7.

Form VII

Form VII is a novel crystalline form of 5-azacytidine. Form VII wasproduced by fast recrystallization from a DMSO/methanol co-solventsystem (see Example 3). Form VII was always isolated by thisrecrystallization method as a mixed phase with Form I. The XRPD powderpattern of mixed phase Forms I and VII is shown in FIG. 7 along withsome of the 2θ values and the Form VII distinctive peaks indicated withasterisks. Table 7 provides the most prominent 2θ angles, d-spacing andrelative intensities for this mixed phase. Form VII exhibits distinctivepeaks at 5.8, 11.5, 12.8, 22.4 and 26.6° 2θ in addition to peaksdisplayed in the Form I XRPD powder pattern. The XRPD pattern for mixedphase Forms I and VII is distinctly different from that of any otherform.

TABLE 7 5-azacytidine, mixed Forms I and VII - the most prominent 2θangles, d-spacing and relative intensities (Cu Ka radiation) 2θ Angle(°) d-spacing (Å) Relative Intensity 5.779 15.281 14.7 11.537 7.664 8.312.208 7.244 28.0 12.759 6.932 21.7 13.048 6.780 34.4 14.418 6.138 22.516.489 5.372 21.6 18.649 4.754 13.5 19.101 4.643 34.7 20.200 4.392 34.420.769 4.273 10.5 21.355 4.157 11.7 22.365 3.972 29.9 23.049 3.856 100.023.884 3.723 23.1 26.628 3.345 13.3 27.145 3.282 52.9 29.296 3.046 26.229.582 3.017 11.3 32.078 2.788 12.9

Thermal analysis of Form VII is presented in Example 6

Form VIII

Form VIII is a novel crystalline form of 5-azacytidine. Form VIII wasrecovered by recrystallizing 5-azacytidine Form I from aN-methyl-2-pyrrolidone (NMP) single solvent system (see Example 4). TheXRPD powder pattern of Form VIII is shown in FIG. 8 along with some ofthe 2θ values. Table 8 provides the most prominent 2θ angles, d-spacingand relative intensities for this material. The XRPD pattern for FormVIII is distinctly different from that of any other form.

TABLE 8 5-azacytidine, Form VIII - the most prominent 2θ angles,d-spacing and relative intensities (Cu Kα radiation) 2θ Angle (°)d-spacing (Å) Relative Intensity 6.599 13.384 2.9 10.660 8.292 2.212.600 7.020 23.4 13.358 6.623 2.6 15.849 5.587 2.0 17.275 5.129 4.220.243 4.383 5.8 20.851 4.257 7.8 21.770 4.079 74.4 22.649 3.923 32.125.554 3.483 100.0 25.740 3.458 7.8 29.293 3.046 3.8 32.148 2.782 8.835.074 2.556 7.4 38.306 2.348 2.5

Amorphous 5-azacytidine

Amorphous 5-azacytidine may be recovered from equilibrium saturatedsolutions of 5-azacytidine in propylene glycol, polyethylene glycol andDMSO. See Example 8.

Pharmaceutical Formulations

For the most effective administration of drug substance of the presentinvention, it is preferred to prepare a pharmaceutical formulation (alsoknown as the “drug product”) preferably in unit dose form, comprisingone or more of the 5-azacytidine forms of the present invention and oneor more pharmaceutically acceptable carrier, diluent, or excipient.

Such pharmaceutical formulation may, without being limited by theteachings set forth herein, include a solid form of the presentinvention which is blended with at least one pharmaceutically acceptableexcipient, diluted by an excipient or enclosed within such a carrierthat can be in the form of a capsule, sachet, tablet, buccal, lozenge,paper, or other container. When the excipient serves as a diluent, itmay be a solid, semi-solid, or liquid material which acts as a vehicle,carrier, or medium for the 5-azacytidine polymorph(s). Thus, theformulations can be in the form of tablets, pills, powders, elixirs,suspensions, emulsions, solutions, syrups, capsules (such as, forexample, soft and hard gelatin capsules), suppositories, sterileinjectable solutions, and sterile packaged powders.

Examples of suitable excipients include, but are not limited to,starches, gum arabic, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include lubricating agents such as, forexample, talc, magnesium stearate and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropyl-hydroxybenzoates; sweetening agents; or flavoring agents.Polyols, buffers, and inert fillers may also be used. Examples ofpolyols include, but are not limited to: mannitol, sorbitol, xylitol,sucrose, maltose, glucose, lactose, dextrose, and the like. Suitablebuffers encompass, but are not limited to, phosphate, citrate, tartrate,succinate, and the like. Other inert fillers which may be used encompassthose which are known in the art and are useful in the manufacture ofvarious dosage forms. If desired, the solid pharmaceutical compositionsmay include other components such as bulling agents and/or granulatingagents, and the like. The compositions of the invention can beformulated so as to provide quick, sustained, controlled, or delayedrelease of the drug substance after administration to the patient byemploying procedures well known in the art.

In certain embodiments of the invention, the 5-azacytidine forms(s) maybe made into the form of dosage units for oral administration. The5-azacytidine forms(s) may be mixed with a solid, pulverant carrier suchas, for example, lactose, saccharose, sorbitol, mannitol, starch,amylopectin, cellulose derivatives or gelatin, as well as with anantifriction agent such as for example, magnesium stearate, calciumstearate, and polyethylene glycol waxes. The mixture is then pressedinto tablets or filled into capsules. If coated tablets, capsules, orpulvules are desired, such tablets, capsules, or pulvules may be coatedwith a concentrated solution of sugar, which may contain gum arabic,gelatin, talc, titanium dioxide, or with a lacquer dissolved in thevolatile organic solvent or mixture of solvents. To this coating,various dyes may be added in order to distinguish among tablets withdifferent active compounds or with different amounts of the activecompound present.

Soft gelatin capsules may be prepared in which capsules contain amixture of the 5-azacytidine form(s) and vegetable oil or non-aqueous,water miscible materials such as, for example, polyethylene glycol andthe like. Hard gelatin capsules may contain granules or powder of the5-azacytidine polymorph in combination with a solid, pulverulentcarrier, such as, for example, lactose, saccharose, sorbitol, mannitol,potato starch, corn starch, amylopectin, cellulose derivatives, orgelatin.

Tablets for oral use are typically prepared in the following manner,although other techniques may be employed. The solid substances aregently ground or sieved to a desired particle size, and a binding agentis homogenized and suspended in a suitable solvent. The 5-azacytidineform(s) and auxiliary agents are mixed with the binding agent solution.The resulting mixture is moistened to form a uniform suspension. Themoistening typically causes the particles to aggregate slightly, and theresulting mass is gently pressed through a stainless steel sieve havinga desired size. The layers of the mixture are then dried in controlleddrying units for a pre-determined length of time to achieve a desiredparticle size and consistency. The granules of the dried mixture aregently sieved to remove any powder. To this mixture, disintegrating,anti-friction, and anti-adhesive agents are added. Finally, the mixtureis pressed into tablets using a machine with the appropriate punches anddies to obtain the desired tablet size.

In the event that the above formulations are to be used for parenteraladministration, such a formulation typically comprises sterile, aqueousand non-aqueous injection solutions comprising one or more 5-azacytidineforms for which preparations are preferably isotonic with the blood ofthe intended recipient. These preparations may contain anti-oxidants,buffers, bacteriostats, and solute; which render the formulationisotonic with the blood of the intended recipient. Aqueous andnon-aqueous suspensions may include suspending agents and thickeningagents. The formulations may be present in unit-dose or multi-dosecontainers, for example, sealed ampules and vials. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets of the kind previously described.

Liquid preparations for oral administration are prepared in the form ofsolutions, syrups, or suspensions with the latter two forms containing,for example, 5-azacytidine polymorph(s), sugar, and a mixture ofethanol, water, glycerol, and propylene glycol. If desired, such liquidpreparations contain coloring agents, flavoring agents, and saccharin.Thickening agents such as carboxymethylcellulose may also be used.

As such, the pharmaceutical formulations of the present invention arepreferably prepared in a unit dosage form, each dosage unit containingfrom about 5 mg to about 200 mg, more usually about 100 mg of the5-azacytidine form(s). In liquid form, dosage unit contains from about 5to about 200 mg. more usually about 100 mg of the 5-azacytidine form(s).The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human subjects/patients or other mammals, eachunit containing a predetermined quantity of the 5-azacytidine polymorphcalculated to produce the desired therapeutic effect, in associationwith preferably, at least one pharmaceutically acceptable carrier,diluent, or excipient.

The following examples are provided for illustrative purposes only, andare not to be construed as limiting the scope of the claims in any way.

EXAMPLES Example 1 Prior Art Procedure for Synthesis of 5-azacytidineDrug Substance

Using commercially available 5-azacytosine (1) and1,2,3,5-Tetra-O-β-acetyl-ribofuranose (2) (RTA), 5-azacytidine (3) maybe synthesized according to the pathway below.

The crude synthesis product is dissolved in DMSO (preheated to about 90°C.), and then methanol is added to the DMSO solution. The co-solventmixture is equilibrated at approximately −20° C. to allow 5-azacytidinecrystal formation. The product is collected by vacuum filtration andallowed to air dry.

Example 2 Slow Recrystallization from DMSO/toluene

Dimethyl sulfoxide (DMSO) was used as the primary solvent to solubilizeForm I of 5-azacytidine and toluene was used as the co-solvent asfollows. Approximately 250 mg of 5-azacytidine was dissolved withapproximately 5 mL of DMSO, preheated to approximately 90° C., inseparate 100-mL beakers. The solids were allowed to dissolve to a clearsolution. Approximately 45 mL of toluene, preheated to approximately 50°C., was added to the solution and the resultant solution was mixed. Thesolution was covered and allowed to equilibrate at ambient conditions.The product was collected by vacuum filtration as white crystals using aBuchner funnel. The collected product was allowed to air dry.

Example 3 Fast Recrystallization from DMSO/methanol, DMSO/toluene, andDMSO/chloroform

Approximately 250 mg of 5-azacytidine was dissolved with approximately 5mL of DMSO as the primary solvent, preheated to approximately 90° C., inseparate 100-mL beakers. The solids were allowed to dissolve to a clearsolution. Approximately 45 mL of the selected co-solvent (methanol,toluene, or chloroform), preheated to approximately 50° C., was added tothe solution and the resultant solution was mixed. The solution wascovered and placed in a freezer to equilibrate at approximately −20° C.to allow crystal formation. Solutions were removed from the freezerafter crystal formation.

The product from the methanol and toluene solutions was collected byvacuum filtration using a Buchner funnel. The resulting whitecrystalline product was allowed to air dry.

The chloroform product was too fine to be collected by vacuumfiltration. Most of the solvent was carefully decanted from thechloroform solution and the solvent from the resultant slurry wasallowed to evaporate at ambient temperature to dryness. The chloroformsolution evaporated to a white product. Note that fast recrystallizationusing the DMSO/methanol co-solvent system has typically been used toprepare 5-azacytidine drug substance in the prior art (see the last stepof the procedure provided in Example 1).

Example 4 Fast Recrystallization N-methyl-2-pyrrolidone (NMP) SingleSolvent System

Approximately 500 mg of 5-azacytidine was dissolved with approximately 5mL of NMP, preheated to approximately 90° C., in separate 50-mL beakers.The solids were allowed to dissolve to a clear solution. The solutionwas covered and placed in a freezer to equilibrate at approximately −20°C. to allow crystal formation. Solutions were removed from the freezerafter crystal formation, equilibrated at ambient temperature. Theproduct was collected by vacuum filtration using a Buchner funnel. Thecollected product was allowed to air dry.

Example 5 X-Ray Powder Diffraction of 5-azacytidine

X-ray powder diffraction patterns for each sample were obtained on aScintag XDS 2000 or a Scintag X₂ θ/θ diffractometer operating withcopper radiation at 45 kV and 40 mA using a Kevex Psi Peltier-cooledsilicon detector or a Thermo ARL Peltier-cooled solid state detector.Source slits of 2 or 4 mm and detector slits of 0.5 or 0.3 mm were usedfor data collection. Recrystallized material was gently milled using anagate mortar and pestle for approximately one minute. Samples wereplaced in a stainless steel or silicon sample holder and leveled using aglass microscope slide. Powder diffraction patterns of the samples wereobtained from 2 to 42° 2θ at 1°/minute. Calibration of the X₂diffractometer is verified annually using a silicon powder standard. Rawdata files were converted to ASCII format, transferred to an IBMcompatible computer and displayed in Origin® 6.1 for Windows.

XRPD of a single sample of 5-azacytidine produced according to themethod of Example 1 revealed that this sample consisted of Form I of5-azacytidine.

NCI retained drug substance samples were also analyzed. These sampleswere all previously synthesized and recrystallized according to themethod of Example 1 and were stored at 5° C. since production. XRPDrevealed some retained samples are comprised of Form I alone, whereasother retained samples contain a mixed phase of Form I and a differentpolymorph, termed Form II.

XRPD of NCI retained drug product samples revealed the existence of FormVI in some samples. In those samples, Form VI was present as a mixedphase with Form I.

XRPD of the recrystallized 5-azacytidine obtained in Example 2 revealedthat slow recrystallization from a DMSO/toluene system produced Form IV.XRPD of the recrystallized 5-azacytidine obtained in Example 3 revealedthat fast recrystallization from a DMSO/chloroform system produced FormN, fast recrystallization from a DMSO/toluene system produced Form V,and fast recrystallization from a DMSO/methanol system produced mixedphased Form I/Form VII. XRPD of the recrystallized 5-azacytidineobtained in Example 4 revealed that the N-methyl-2-pyrrolidone solventsystem produced Form VIII.

Example 6 Thermal Analysis of 5-azacytidine

Differential Scanning calorimetry (DSC) measurements for each samplewere collected using a Perkin Elmer Pyris 1 DSC system equipped with anIntracooler 2P refrigeration unit. The Pyris 1 DSC was purged withnitrogen. Calibration was performed prior to analysis using an Indiumstandard at a 10° C. minute heating rate. Each sample was gently groundin an agate mortar and pestle. Approximately 1-3 mg of the sample wereindividually sealed in a Perkin Elmer 30-μL universal aluminum pan withholes in the lid. Samples were heated from 25° C. to 250° C. or 350° C.at 10° C./minute.

Thermogravimetric Analysis (TGA) measurements for each sample werecollected using a Perkin Elmer TGA 7 purged with nitrogen atapproximately 20 cc/minute. A 100-mg standard weight and nickel metalwere used to verify balance and temperature calibrations, respectively.Samples were heated from 25° C. to 250° C. or 300° C. at 10° C./minute.

Capillary melting point (MP) measurements were made using anElectrothermal 9300 melting point apparatus. A heating rate of 10°C./minute was used from set point temperatures described in individualdiscussions. Visual melting points are reported as an average oftriplicate determinations.

The results are as follows:

Form I

TGA showed a weight loss of 0.23% between ambient and 150° C., whichindicates that it is anhydrous. DSC exhibited a single event with anonset of 227.0° C.

A capillary melting point determination was performed in triplicate on asample of Form I of 5-azacytidine. The sample was visually observed todecompose without melting at about 215° C. using a 10° C. heating rateand a starting temperature of 200° C. Thus, the DSC event results fromdecomposition of 5-azacytidine.

Form I/II Mixed Phase

The TGA for the Form I/II mixed phase showed a weight loss of 1.16%between ambient temperature and 150° C. The DSC analysis exhibited asingle event with an onset at 229.8° C. The decomposition of the mixedphase was consistent with that observed for 5-azacytidine Form I.

Form III

The TGA showed a weight loss of between 6.56% and 8.44% when thetemperature was raised from ambient and 150° C. The loss is close to thetheoretical amount of moisture, 6.9%, that 5-azacytidine monohydratewould have. The DSC analysis exhibited an endotherm, which is in therange associated with solvent loss, and a higher temperature event. Theendotherm exhibited an onset temperature in the range of 86.4-89.2° C.,peak temperatures in the range of 95.8-97.0° C. and ΔH values in therange of 73.1-100.5 J/g. The higher temperature event had onsettemperatures in the range 229.1-232.1° C. and was consistent with thedecomposition observed for 5-azacytidine Form I.

5-azacytidine Form III was heated at 105° C. for 4 hours in an attemptto dehydrate the material. The material did not change its physicalappearance during heating. TGA was used to measure the water content ofForm III before and after drying. The initial amount of moisture presentin Form III was 6.31% and was <0.1% after drying. The XRPD powderpattern for dehydrated Form III matches that of Form I. Thus, Form IIIdehydrates to Form I.

Form IV

The TGA showed a weight loss of 21.80% between ambient temperature and150° C., which does not correspond to the solvent content for any simplesolvates. It is not known whether crystalline Form IV is a polymorph ora pseudopolymorph.

The DSC analysis exhibited two endotherms and a higher temperatureevent. The two endotherms are in the range that is associated withsolvent loss. The first endotherm exhibited an onset temperature of87.6° C., a peak temperature of 90.1° C. and ΔH value of 98.3 J/g. Thesecond endotherm exhibited an onset temperature of 136.0° C., a peaktemperature of 139.0° C. and ΔH value of 81.8 J/g. The highertemperature event had an onset temperature of 230.6° C. and wasconsistent with the decomposition that was observed for 5-azacytidineForm I.

Form V

TG A showed a weight loss of 21.45% between ambient and 150° C., whichdoes not correspond to the solvent content for any simple solvate. TheDSC analysis exhibited two merged endotherms, a single endotherm and ahigher temperature event. The three endotherms are in the range that isassociated with solvent loss. The two merged endotherms exhibit onsettemperatures of 66.6 and 68.0° C. The single endotherm exhibited anonset temperature of 88.7° C., a peak temperature of 121.5° C. and a ΔHvalue of 180.3 μg. The higher temperature event had onset temperature of230.7° C. and was consistent with the decomposition that was observedfor 5-azacytidine Form I.

Form VI

TGA showed a weight loss of 1.10% between ambient temperature and 150°C. The DSC analysis exhibited a small endotherm, an exotherm and ahigher temperature event. The small endotherm exhibited an onsettemperature of 57.8° C., a peak temperature of 77.0° C. and a ΔH valueof 55.7 J/g. The exotherm exhibited an onset temperature of 149.3° C., apeak temperature of 157.1° C. and an ΔH value of −17.9 J/g. The highertemperature event had an onset temperature of 234.7° C. and wasconsistent with the decomposition observed for 5-azacytidine Form I.

Form VII

TGA showed a weight loss of 2.45% between ambient temperature and 150°C. The DSC analysis exhibited a minor endotherm and a higher temperatureevent. The minor endotherm had an onset temperature of 63.3° C., a peaktemperature of 68.3° C. and a ΔH value of 17.1 J/g. The highertemperature event had an onset temperature of 227.2° C. and isconsistent with the decomposition observed for 5-azacytidine Form I.

Example 7 Nuclear Magnetic Resonance (NMR) Analysis of Form III and FormVI

5-azacytidine is know to be labile in water. Since Form III is found inequilibrium saturated solutions and Form VI is produced by thelyophilization of 5-azacytidine solution. it was of interest to examinethe purity of these 5-azacytidine forms using NMR. The proton (¹H) NMRspectra of Form III and Form VI were both consistent with the structureof 5-azacytidine in all essential details.

Example 8 Polymorphic Form Conversion of 5-azacytidine

Form I of 5-azacytidine was added to various solvents in sufficientquantities to form a slurry, and the slurry allowed to equilibrate for aperiod of time. The solid material that was present in the slurry wasrecovered, dried, and analyzed using XRPD (according to the XRPDprotocol included in Example 5) with the aim of detecting new polymorphsand pseudopolymorphs during the transition to the dissolved state.Samples equilibrated for 19 hours in saline, 5% dextrose, 5% tween 80,water-saturated octanol, ethanol/water (50/50) and water alone resultedin a distinctly different form of 5-azacytidine, designated Form III(see below). Samples equilibrated for 19 hours in acetone, methyl ethylketone, and ethanol resulted in materials identified as Form I. Samplesequilibrated for 19 hours in propylene glycol, polyethylene glycol andDMSO resulted in amorphous materials. The results are summarized inTable 9.

TABLE 9 X-ray Powder Diffraction Analysis Results for SolubilitySamples: Form Assignment (Cu Kα radiation) XRPD Pattern SolventAssignment Normal Saline Form III 5% Dextrose Form III Acetone Form IPropylene glycol Amorphous Polyethylene glycol Amorphous Methyl ethylketone Form I 5% Tween 80 Form III DMSO Amorphous Water-saturatedOctanol Form III Ethyl alcohol Form I 50/50 EtOH/DI Water Form III DIWater Form III

The conversion of other forms of 5-azacytidine was also studied.Specifically, a Form I/II mixed phase, Form VI (the lyophilized drugproduct used in the prior art NCI drug trials), a Form I/VI mixed phase,and a Form I/VII mixed phase were weighed into individual small glassbeakers and water was pipetted into each beaker. The sample size andwater volume were scaled to maintain an approximate 25 mg/mL ratio. Theresultant slurry was allowed to equilibrate for 15 minutes. Afterequilibration, the sample was filtered and the solid material was driedand analyzed using XRPD. In each case, Form III of 5-azacytidine wasobserved. The results indicate that all forms of 5-azacytidine convertto Form III during the transition to the dissolved state in water. Thus,when an 5-azacytidine suspension (“slurry”) was administered to patientsin the aforementioned NCI investigation, the patients received both5-azacytidine in solution, and Form III of 5-azacytidine.

1-13. (canceled)
 14. A pharmaceutical composition for oral administration comprising Form I and Form VII of 5-azacytidine substantially free of other forms of 5-azacytidine.
 15. The pharmaceutical composition of claim 14, wherein the Form VII of 5-azacytidine is characterized by X-Ray Powder Diffraction peaks at approximately 5.8, 11.5, 12.8, 22.4, and 26.6 degrees 2θ.
 16. The pharmaceutical composition of claim 14, wherein the Form I and Form VII of 5-azacytidine is characterized by X-Ray Powder Diffraction peaks at the following 2θ angles: 2θ Angle (degree) 5.779 11.537 12.208 12.759 13.048 14.418 16.489 18.649 19.101 20.200 20.769 21.355 22.365 23.049 23.884 26.628 27.145 29.296 29.582 32.078


17. The pharmaceutical composition of claim 14, wherein the Form I and Form VII of 5-azacytidine is characterized by an X-ray powder diffraction pattern corresponding to the representative X-Ray Powder Diffraction Pattern provided in FIG.
 7. 18. The pharmaceutical composition of claim 14, wherein the Form I and Form VII of 5-azacytidine has a thermogravimetric analysis thermogram comprising a weight loss of about 2.45% between ambient temperature and 150° C.
 19. The pharmaceutical composition of claim 14, wherein the Form I and Form VII of 5-azacytidine has a differential scanning calorimetry thermogram comprising an endotherm with a peak temperature of about 68.3° C.
 20. The pharmaceutical composition of claim 14, which is in a single unit dosage form.
 21. The pharmaceutical composition of claim 14, which is a tablet.
 22. The pharmaceutical composition of claim 14, which is a capsule.
 23. The pharmaceutical composition of claim 14, which contains between about 5 mg and about 200 mg of 5-azacytidine.
 24. The pharmaceutical composition of claim 23, which contains about 100 mg of 5-azacytidine.
 25. The pharmaceutical composition of claim 14, which further comprises a carrier, diluent, or excipient.
 26. The pharmaceutical composition of claim 25, which comprises at least one of mannitol, microcrystalline cellulose, and magnesium stearate. 